We have compared the functional properties of Cgamma4+ hybridomas derived from newborn thymocytes and from adult spleens with Cgamma4+ dendritic in epidermal T cells (DETC) in terms of their structural features, ability to secrete cytokines spontaneously and for the use of the vitronectin receptor (VNR) as an accessory molecule. The thymic hybridomas secreted lymphokines spontaneously and were dependent on VNR for this activity whereas the splenic hybridomas failed to secrete lymphokines. Five thymic hybrids expressed the Vgamma1.1Cgamma4/Vk6 receptor and one hybrid a Vgamma1.1Cgamma4/Vk4 receptor that had a close structural relationship to the DETC gamma/delta TcR associated with spontaneous lymphokine secretion. The strong correlation of functional and molecular properties between thymocyte Cgamma4+ hybridomas and Cgamma4+ DETC suggests that the Cgamma4+ DETC may be of thymic origin and that cells with potential for autoreactivity residing in the thymus at birth may populate other peripheral locations in the mouse. To further evaluate the role of the TcR, the VNR and the putative autoantigen in the activation of the Cgamma4+ T cell subset, we cloned complete cDNA encoding the Vgamma1.1Cgamma4 and Vdelta6Cdelta TcR and transfected the cDNA constructs into a TcR- murine hybridoma and into a TcR- variant of the human Jurkat line. The murine transfectant spontaneously produced IL-2 in culture and IL-2 production could be inhibited by anti-CD3, anticlonotypic mAb to the transfected TcR, and anti-VNR mAb, as well as by RGDS. These results demonstrated that transfection of the gamma delta TcR confers to recipient T cells the phenotype of constitutive activation, as well as dependence on engagement of the VNR as an accessory molecule. In contrast, the Jurkat gamma delta transfectant failed to produce cytokines spontaneously. Surprisingly, neither the murine transfectant nor the human transfectant could be induced to respond to autoantigen bearing cells in coculture assays. One interpretation of these results is that coexpression on the surface of the same cell of the Vgamma1.1Cgamma4Vdelta6 TcR, the VNR, and a putative autoantigen are necessary for the T cell activation in this system. Integrins are a superfamily of alpha beta heterodimers, most of which serve as cell surface receptors for extracellular matrix proteins. We demonstrated that the recently described alpha6beta4 integrin, previously thought to be limited to epithelial cells and Schwann cells, is expressed on immature mouse thymocytes. The presence of alpha6beta4 is controlled by regulation of betaa4 expression. During fetal ontogeny, beta4 was highly expressed by day-13 to day 15 thymocytes, then rapidly declined to low levels by birth. In neonates and adults, beta4 expression was highest on CD4- CD8- CD3- and TcR+- gamma delta subsets. Overall, down-regulation of beta4 was associated with up-regulation of CD4, CD8, and CD3 in the thymus. The data suggest that alpha6beta4 is up-regulated after pro-T cells enter the thymus and may have a thymus-specific function for T cells.